Project Summary The epidermis provides protection from environmental insult, dehydration and stress. Mechanical strength is derived from robust cell-cell adhesive junctions called desmosomes is a fundamental feature of epidermal tissue. Desmosomes are macromolecular complexes composed of desmosomal cadherins, which mediate cell- cell adhesion, and a number of intracellular plaque proteins, including desmoplakin, which couples the complex to the intermediate filament cytoskeleton. Notably, aberrant desmosome function can lead to severe epidermal disorders. Pemphigus vulgaris is a potentially life-threatening skin blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3) that leads to disruption of cell-cell adhesion. Though responsible for mechanical integrity, desmosomes can switch between strong and weak states in development and wound healing. This functional transition occurs with minimal change to the core proteins comprising the desmosome. We hypothesize that the architecture or organization of proteins within a desmosome drives its adhesive function. However, due to the size and molecular complexity of desmosomes there is a lack of tools to study this structure-function relationship creating a critical barrier in this field. We will use a multi-disciplinary approach to address this challenge and to test the hypothesis that the biophysical organization of proteins in the desmosome provides a mechanism to regulate adhesion. We recently developed two highly innovative and complimentary super-resolution fluorescence microscopy approaches to study the order and organization of proteins within desmosomes. Our goal is to elucidate how the order and organization of proteins impacts the adhesive function of desmosomes in healthy and disease states. This will provide novel insight into the structure and function of these critical complexes. In Aim 1 we will determine the how the organization of plaque proteins changes in different adhesive states with the goal of identifying functionally sensitive elements and potential biomarkers. In Aim 2 we will use a live cell approach to study mechanisms that confer ordering of desmosomal cadherins, and how this order is altered with function. Finally, in Aim 3 we will define changes to the architecture of the desmosome induced in pemphigus vulgaris, with the goal of facilitating development of targeted therapeutics. We will use primary human keratinocytes and human tissue biopsy samples to address these questions. Accomplishment of these goals will provide a fundamental understanding and framework of how protein organization and dynamics influence the adhesive function of desmosomes in healthy and disease states.